The saltwater mosquito larva, Aedes dorsal is, is one of the only
organisms capable of inhabiting hypersaline lakes composed almost entirely of
high concentrations of NaHC03 and Na2C03 salts. Under laboratory conditions
larvae survived and developed normally in saline media with pH values up to
10.5, HC0₃⁻ concentrations up to 250 mM, or C0₃²- concentrations up to
100 mM. Despite ingestion of these alkaline media at rates equivalent to
130% of larval body weight per day, these insects regulated hemolymph pH
(7.55-7.70) and HCO₃⁻ concentrations (8.0 - 18.5 mM) within narrow
physiological limits. Micropuncture and microcannulation studies on the
rectal salt gland demonstrated that this organ was an important site of pH
and HC0₃⁻ regulation. Microcannulated salt glands secreted a strongly
hyperosmotic fluid containing 402 mM HCO₃⁻ and 41 mM C0₃²⁻ at a rate of
38 nl-h⁻¹. Lumen-to-bath HC0₃⁻ and C0₃²⁻ gradients of 21:1 and 241:1,
respectively, were generated by the salt gland epithelium against a
transepithelial potential of -25 mV (lumen negative) demonstrating clearly
the active nature of HCO₃⁻ secretion.
To study the mechanisms of HCO₃⁻ transport, an in vitro
microperfused rectal salt gland preparation was developed. Net total CO₂
transport (J[sup=JCo₂; sub=net]) as measured by microcalorimetry in perfused salt glands was
unaffected by bilateral Na⁺ or K⁺ and serosal Cl⁻ substitutions, or by
serosal addition of 1.0 mM ouabain, 2.0 mM amiloride or 0.5 mM SITS.
Removal of luminal Cl⁻ inhibited (J[sup=JCo₂; sub=net] by 80%, while serosal addition of 1.0 mM acetazolamide or 0.5 mM DIDS inhibited by (J[sup=JCo₂; sub=net] 80% and 40%, respectively.
Perfusion of the anterior and posterior rectal segments demonstrated
clearly that the anterior rectum was the site of CO₂ secretion in
the microperfused salt gland. Net Cl⁻ reabsorption in the anterior segment was measured by electron microprobe analysis and was equivalent to the rate of CO₂ secretion. In addition, Cl⁻ reabsorption in the anterior segment was completely inhibited by bilaterally replacing C0₂ and HC0₃⁻ with a phosphate or HEPES buffered saline. These data provide strong quantitative evidence for the presence of a 1:1 Cl⁻/HCO₃⁻ exchange mechanism located in the anterior rectal salt gland segment.
Microcannulation studies on the individual salt gland segments demonstrated that both rectal segments are capable of secreting a hyperosmotic fluid containing Na⁻, Cl⁻ and HCO₃⁻. Based on these results and the results of studies in which the effects of serosal ion substitutions on salt gland fluid secretion were examined, it has been suggested tentatively that both segments secrete a NaCl-rich fluid and that fluid secretion is driven by coupled NaCl transport. It is further suggested that once this fluid enters the salt gland lumen its composition is modified by ion exchange and reabsorptive processes which are dependent upon the ionic regulatory needs of the animal. In larvae inhabiting low Cl⁻, NaHCO₃-CO₃ lakes, this modification involves a 1:1 exchange of luminal Cl⁻ for serosal HCO₃⁻.
The cellular mechanisms of anterior salt gland HCO₃⁻ and Cl⁻ transport were examined using ion and voltage-selective microelectrodes in conjunction with a microperfused anterior segment preparation which allowed complete changes in serosal and mucosal saline composition to be made in <5-10 seconds. Addition of DIDS or acetazolamide to or removal of CO₂ and HCO₃⁻ from the serosal bath caused large, 20-50 mV hyperpolarizations of Va and had little effect on . Rapid changes in luminal Cl⁻ concentration altered Va in a rapid, step-wise manner. The slope of the
relationship between Va and luminal Cl⁻ activity was 42.2 mV/decalog a[sup=l; sub=Cl⁻ (r = 0.992). Intracellular Cl⁻ activity was 23.5 mM and was approximately 10 mM lower than that predicted for a passive distribution at the apical membrane. Changes in serosal Cl⁻ concentration had no effect on indicating an electrically silent basolateral Cl⁻ exit step. Intracellular
pH in anterior rectal cells was 7.67 and the calculated a[sup=C;sub=HCO₃] - was 14.4 mM.
These results show that under control conditions HC0₃⁻ enters the anterior rectal cell by an active mechanism against an electrochemical gradient of 77.1 mV and exits the cell at the apical membrane down a favorable electrochemical gradient of 27.6 mV. Based on these results, a tentative cellular model has been proposed in which Cl⁻ enters the apical membrane of the anterior rectal cells by passive, electrodiffusive movement through a Cl⁻-selective channel, and HCO₃⁻ exits the cell by an active or passive electrogenic transport mechanism. The electrically silent nature of
basolateral Cl⁻ exit and HC0₃⁻ entry, and the effects of serosal addition of Cl⁻/HCO₃⁻ exchange inhibitor DIDS on (J[sup=JCo₂; sub=net] and V[sub=te] suggest strongly that
the basolateral membrane is the site of a direct coupling between Cl⁻ and
HCO₃⁻ movements via a Cl⁻/HC0₃⁻ exchange mechanism. / Science, Faculty of / Zoology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/24602 |
| Date | January 1983 |
| Creators | Strange, Kevin |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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